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Povzetek: The giant colonial ciliate Zoothamnium niveum harbors a monolayer of the gammaproteobacteria Cand. Thiobios zoothamnicoli on its outer surface. Cultivation experiments revealed maximal growth and survival under steady flow of high oxygen and low sulfide concentrations. We aimed at directly demonstrating the sulfur-oxidizing, chemoautotrophic nature of the symbionts and at investigating putative carbon transfer from the symbiont to the ciliate host. We performed pulse-chase incubations with 14C- and 13C-labeled bicarbonate under varying environmental conditions. A combination of tissue autoradiography and nanoscale secondary ion mass spectrometry coupled with transmission electron microscopy was used to follow the fate of the radioactive and stable isotopes of carbon, respectively. We show that symbiont cells fix substantial amounts of inorganic carbon in the presence of sulfide, but also (to a lesser degree) in the absence of sulfide by utilizing internally stored sulfur. Isotope labeling patterns point to translocation of organic carbon to the host through both release of these compounds and digestion of symbiont cells. The latter mechanism is also supported by ultracytochemical detection of acid phosphatase in lysosomes and in food vacuoles of ciliate cells. Fluorescence in situ hybridization of freshly collected ciliates revealed that the vast majority of ingested microbial cells were ectosymbionts.
Ključne besede: microbial ecology, symbiosis
Objavljeno v DiRROS: 24.07.2024; Ogledov: 234; Prenosov: 207
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Povzetek: Cloning multiple DNA fragments for delivery of several genes of interest into the plant genome is one of the main technological challenges in plant synthetic biology. Despite several modular assembly methods developed in recent years, the plant biotechnology community has not widely adopted them yet, probably due to the lack of appropriate vectors and software tools. Here we present Plant X-tender, an extension of the highly efficient, scar-free and sequence-independent multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for most efficient cloning into the novel vector set needed for plant expression and thus introduces advantages of AssemblX into plant synthetic biology. The novel vector set covers different backbones and selection markers to allow full design flexibility. We have included ccdB counterselection, thereby allowing the transfer of multigene constructs into the novel vector set in a straightforward and highly efficient way. Vectors are available as empty backbones and are fully flexible regarding the orientation of expression cassettes and addition of linkers between them, if required. We optimised the assembly and subcloning protocol by testing different scar-less assembly approaches: the noncommercial SLiCE and TAR methods and the commercial Gibson assembly and NEBuilder HiFi DNA assembly kits. Plant X-tender was applicable even in combination with low efficient homemade chemically competent or electrocompetent Escherichia coli. We have further validated the developed procedure for plant protein expression by cloning two cassettes into the newly developed vectors and subsequently transferred them to Nicotiana benthamiana in a transient expression setup. Thereby we show that multigene constructs can be delivered into plant cells in a streamlined and highly efficient way. Our results will support faster introduction of synthetic biology into plant science.
Ključne besede: cloning, plasmid construction, polymerase chain reaction
Objavljeno v DiRROS: 24.07.2024; Ogledov: 288; Prenosov: 173
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Povzetek: Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection.
Ključne besede: digital PCR, droplet digital PCR, absolute quantification, reference materials, GMO quantification
Objavljeno v DiRROS: 24.07.2024; Ogledov: 391; Prenosov: 145
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Povzetek: Bois noir (BN) is the most widespread European grapevine yellows disease caused by ‘Candidatus Phytoplasma solani’. Although our knowledge of the mechanisms of interactions of this pathogenic bacteria with host is largely unknown, the plant-pathogen system of BN is commonly used as a model system for studying grapevine yellows diseases. We applied here a conceptual model of general plant pathology – a disease triangle for describing interactions among the host plant, the pathogen and the environment. We generated a proof-of-concept statistical model for disease triangle using original experimental data and different statistical and data mining approaches for a selected system of ‘Ca. P. solani’ infection of cv. ‘Chardonnay’ grapevine plants. We monitored individual plants from a single vineyard over a period of six years. Phytoplasma content, the expression of 21 selected grapevine genes and environmental conditions were recorded and related to disease severity. Our model predicts that in described conditions BN is a function of the expression of grapevine gene VvDMR6, summer rainfall and abundance of ‘Ca. P. solani’. The greatest impact among elements of the disease triangle is attributed to the pathogen, and is independent of the pathogen titer. We showed that this first de facto representation of the disease triangle is useful for showing disease dynamics over several years and could be applied to other plant-pathogen systems. The overall results of this study will contribute to understanding of ‘Ca. P. solani’ biology and its interactions with grapevine host.
Ključne besede: plant diseases, plant pathology
Objavljeno v DiRROS: 24.07.2024; Ogledov: 464; Prenosov: 239
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Povzetek: Xanthomonas oryzae pv. oryzicola (Xoc) is a seed-borne bacterial pathogen of rice. In this study, a rapid molecular detection method in seeds was developed to prevent unwanted movement of infected materials and greater yield loss. Crude DNA from ground seed extract supernatant was precipitated in ethanol and centrifuged. DNA pellets of homogenous subsamples were pooled and werepurified using SDS or a DNA extraction kit. Three pairs of primers previously designed on Xoc isolate BLS-256 genome sequence robustly amplify Xoc-specific DNA sequences. Primer T40 was found to efficiently detect Xoc but only in single primer reactions. While primers 3864 and 3866 can amplify Xoc DNA separately, however, their multiplex PCR reactions were unsuccessful, probably due to substrate competition in the reaction mixture. Optimized PCR mixture and conditions were able to detect as low as 10 cfu/mL Xoc cells and the direct extraction method and conditions were able to detect as low as 104 cfu/mL inoculated cells using primer T40. Lastly, Xoc transmission through seeds was observed only in plant samples which were heavily infected. Recovery from infection, at around 25-50% was also observed in some cultivars which also manifested good grain filling at 56 days after transplanting.
Objavljeno v DiRROS: 24.07.2024; Ogledov: 225; Prenosov: 136
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